Platelets promote the catalysis of two sequential calcium-dependent reactions in blood coagulation: the activation of factor X (FX) by a complex of FIXa and FVIIIa and the conversion of prothrombin to thrombin by a complex of FXa and FVa. The contribution of platelets to F-X activation is receptor-mediated since platelets possess specific, high- affinity, saturable binding sites for FIXa and FVIII and receptor occupancy is closely correlated with rates of F-X activation on the platelet surface. Our recent studies have demonstrated that activated human platelets expose 500-600 FIXa binding sites per platelet with a K/d(app) of about 2.5 nM in the absence of FVIII and FX and the same number of sites with enhanced affinity (K/d(app) of about 0.5 nM) in the presence of FVIII and FX. We have also confirmed the observation of Nesheim and his colleagues who have demonstrated the presence of a single class of binding sites (450/platelet, K/d = 2.9 nM) for recombinant human FVIII (rFVIII) on thrombin-activated human platelets. Moreover, we have demonstrated the presence of a low-affinity, high capacity binding site on activated human platelets for FX which is shared with prothrombin and a lower capacity, higher affinity site that is specific for FX in the presence of FIXa and FVIII. These observations support the hypothesis that the F-X activating complex on the platelet surface consists of a three-receptor complex, the assembly of which results in a 24 million-fold acceleration of the rate of F-X activation. The purpose of the studies proposed in this application is to examine in more detail the validity of this hypothesis and to determine the structural components on the platelet surface and on the enzyme (FIXa) required for the assembly of this important coagulation complex. Specifically, we propose to accomplish a complete characterization of the F-X activating complex on the platelet surface by carrying out coordinate binding studies with FIXa, FVIII(a), and FX and simultaneous kinetic studies of F-X activation. We propose to determine the structural domains in FIXa required for binding to its platelet receptor and for assembly of the F-X activating complex, specifically focusing upon the role of the Gla domain and the EGF domains. We will determine the state of platelet activation required for binding the components of the F-X activating complex and carry out studies aimed to determine the subcellular localization and biochemical characterization of the platelet receptors essential for binding the components of the F-X activating complex.